Journal of cytokine biology最新文献

{"title":"Chemical Modification of CRISPR <i>gRNAs</i> Eliminate type I Interferon Responses in Human Peripheral Blood Mononuclear Cells.","authors":"Mollie S Schubert, Edward Cedrone, Barry Neun, Mark A Behlke, Marina A Dobrovolskaia","doi":"10.4172/2576-3881.1000121","DOIUrl":"10.4172/2576-3881.1000121","url":null,"abstract":"<p><strong>Objectives: </strong>CRISPR/Cas9 is currently the primary tool used for genome editing in mammalian cells. To cleave and alter genomic DNA, both the Cas9 nuclease and a guide RNA (<i>gRNA</i>) must be present in the nucleus. One preferred method of introducing these reagents is direct transfection of a recombinant Cas9 protein complexed with a synthetic <i>gRNA</i> as a ribonucleoprotein (RNP) complex. It is well established from prior work in RNA interference that synthetic RNAs can induce a type I interferon (IFN) response that can limit the application of such methods both <i>in vitro</i> and <i>in vivo.</i> While the immunological properties of short siRNAs are well understood, little is known about the immune recognition of longer CRISPR <i>gRNAs.</i> The objective of our <i>in vitro</i> study was to investigate how the composition of the <i>gRNA</i> influences its recognition by human immune cells.</p><p><strong>Methods: </strong>The study was performed <i>in vitro</i> in human peripheral blood mononuclear cells (PBMCs). The PBMCs from healthy donor volunteers were treated with <i>gRNA</i> for 24 h, and the levels of type I IFNs in culture supernatants were measured by a multiplex enzyme-linked immunosorbent chemiluminescent assay. Prior to the analysis in PBMCs, the physicochemical parameters and functionality of all nucleic acid constructs were confirmed by electrospray-ionization mass spectrometry and CRISPR/Cas9 gene editing assessment in HEK293-Cas9 cells, respectively.</p><p><strong>Results: </strong>We found that unmodified synthetic CRISPR <i>gRNAs</i> triggered a strong IFN response in PBMC cultures <i>in vitro</i> that could be prevented with chemical modification. Likewise, <i>in vitro</i>-transcribed single-guide RNAs (<i>sgRNAs</i>) also triggered a strong IFN response that could only be partially suppressed by phosphatase removal of the 5'-triphosphate group. However, the process by which the <i>gRNA</i> is prepared (i.e., chemically synthesized as a two-part <i>crRNA:tracrRNA</i> complex or <i>in vitro</i>-transcribed as an <i>sgRNA</i>) does not directly influence the immune response to an unmodified <i>gRNA.</i> When experiments were performed in the HEK293 cells, only <i>in vitro</i>-transcribed <i>sgRNA</i> containing 5'-triphosphate induced IFN secretion.</p><p><strong>Conclusion: </strong>The results of our structure-activity relationship study, therefore, suggest that chemical modifications commonly used to reduce the immunostimulation of traditional RNA therapeutics can also be used as effective tools to eliminate undesirable IFN responses to <i>gRNAs.</i></p>","PeriodicalId":91902,"journal":{"name":"Journal of cytokine biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/7d/06/nihms944678.PMC6138052.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36499646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cytokine and Lipid Mediator Regulation of Group 2 Innate Lymphoid Cells (ILC2s) in Human Allergic Airway Disease.","authors":"Kellen Cavagnero,&nbsp;Taylor A Doherty","doi":"10.4172/2576-3881.1000116","DOIUrl":"https://doi.org/10.4172/2576-3881.1000116","url":null,"abstract":"<p><p>The recent discovery of group 2 innate lymphoid cells (ILC2s) has caused a paradigm shift in the understanding of allergic airway disease pathogenesis. Prior to the discovery of ILC2s, Th2 cells were largely thought to be the primary source of type 2 cytokines; however, activated ILC2s have since been shown to contribute significantly, and in some cases, dominantly to type 2 cytokine production. Since the discovery of ILC2s in 2010, many mediators have been shown to regulate their effector functions. Initial studies identified the epithelial derived cytokines IL-25, IL-33, and TSLP as activators of ILC2s, and recent studies have identified many additional cytokine and lipid mediators that are involved in ILC2 regulation. ILC2s and their mediators represent novel therapeutic targets for allergic airway diseases and intensive investigation is underway to better understand ILC2 biology and upstream and downstream pathways that lead to ILC2-driven airway pathology. In this review, we will focus on the cytokine and lipid mediators that regulate ILC2s in human allergic airway disease, as well as highlight newly discovered mediators of mouse ILC2s that may eventually translate to humans.</p>","PeriodicalId":91902,"journal":{"name":"Journal of cytokine biology","volume":"2 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4172/2576-3881.1000116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35397539","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impacts of Interleukin-17 Neutralization on the Inflammatory Response in a Healing Ligament.","authors":"Anna Eb Clements,&nbsp;Connie S Chamberlain,&nbsp;Ellen M Leiferman,&nbsp;William L Murphy,&nbsp;Ray Vanderby","doi":"10.4172/2576-3881.1000113","DOIUrl":"https://doi.org/10.4172/2576-3881.1000113","url":null,"abstract":"<p><p>In this study, we sought to improve ligament healing by modulating the inflammatory response after acute injury through the neutralization of Interleukin-17 (IL-17), which we hypothesized would decrease inflammatory cell infiltration and cytokine production. Administration of an Interleukin-17 neutralizing antibody (IL-17 NA) immediately following a rat medial collateral ligament (MCL) transection resulted in alterations in inflammatory cell populations and cytokine expression within the healing ligament, but did not reduce inflammation. Specifically, treatment resulted in a decrease in M2 (anti-inflammatory) macrophages, an increase in T cells, and an increase in the levels of IL-2, IL-6, and IL-12 in the MCL 7 days post injury. IL-17NA treatment, and subsequent immunomodulation, did not result in improved ligament healing, as measured by collagen composition and wound size.</p>","PeriodicalId":91902,"journal":{"name":"Journal of cytokine biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4172/2576-3881.1000113","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35296403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mesenchymal Stem Cell Therapy on Tendon/Ligament Healing.","authors":"Connie S Chamberlain,&nbsp;Erin E Saether,&nbsp;Erdem Aktas,&nbsp;Ray Vanderby","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>A normal healing response after ligament and tendon rupture results in scar formation and an inferior tissue that fails to emulate its original structure, composition, and function. More regenerative healing (closer to the original) can be obtained through early suppression of inflammatory cells and associated cytokines. Examination of the immune mediated response of mesenchymal stem/stromal cells (MSCs) during healing indicates that MSCs reprogram macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Based on these studies our objective was to treat ligament and tendon injuries with MSCs in order to modulate their inflammatory response. Our initial studies using allogeneic cells demonstrated an <i>in vivo</i> dose dependency of MSCs on ligament healing. Medial collateral ligaments (MCLs) treated with 1 × 10⁶ (low dose) MSCs exhibited less inflammation and a reduced number of M1 macrophages compared to ligaments treated with 4 × 10⁶ (high dose) MSCs. Strength of ligament was also improved with the low dose treatment. We then examined the <i>in vivo</i> effects of MSCs that had been preconditioned to be more anti-inflammatory. Treatment with these preconditioned MSCs was compared with normally processed (unconditioned) MSCs using the rat Achilles tendon and MCL healing models. Pre-conditioned MSCs significantly reduced inflammation by increasing the M2 macrophages and decreasing the M1 macrophages. Most importantly, treatment with pre-conditioned MSCs improved tissue strength to levels comparable to intact tissue. Overall, pre-conditioned MSC-treatment out-performed unconditioned MSCs to improve ligament and tendon healing by stimulating a more robust, paracrine-mediated immunosuppressive response.</p>","PeriodicalId":91902,"journal":{"name":"Journal of cytokine biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493432/pdf/nihms864425.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35136957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mesenchymal Stem Cell Therapy on Tendon/Ligament Healing.","authors":"C. Chamberlain, Erin E. Saether, E. Aktaş, R. Vanderby","doi":"10.4172/2576-3881.1000112","DOIUrl":"https://doi.org/10.4172/2576-3881.1000112","url":null,"abstract":"A normal healing response after ligament and tendon rupture results in scar formation and an inferior tissue that fails to emulate its original structure, composition, and function. More regenerative healing (closer to the original) can be obtained through early suppression of inflammatory cells and associated cytokines. Examination of the immune mediated response of mesenchymal stem/stromal cells (MSCs) during healing indicates that MSCs reprogram macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Based on these studies our objective was to treat ligament and tendon injuries with MSCs in order to modulate their inflammatory response. Our initial studies using allogeneic cells demonstrated an in vivo dose dependency of MSCs on ligament healing. Medial collateral ligaments (MCLs) treated with 1 × 106 (low dose) MSCs exhibited less inflammation and a reduced number of M1 macrophages compared to ligaments treated with 4 × 106 (high dose) MSCs. Strength of ligament was also improved with the low dose treatment. We then examined the in vivo effects of MSCs that had been preconditioned to be more anti-inflammatory. Treatment with these preconditioned MSCs was compared with normally processed (unconditioned) MSCs using the rat Achilles tendon and MCL healing models. Pre-conditioned MSCs significantly reduced inflammation by increasing the M2 macrophages and decreasing the M1 macrophages. Most importantly, treatment with pre-conditioned MSCs improved tissue strength to levels comparable to intact tissue. Overall, pre-conditioned MSC-treatment out-performed unconditioned MSCs to improve ligament and tendon healing by stimulating a more robust, paracrine-mediated immunosuppressive response.","PeriodicalId":91902,"journal":{"name":"Journal of cytokine biology","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85486722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}